Auxin polar transport is essential for the development of zygote and embryo in Nicotiana tabacum L. and correlated with ABP1 and PM H+-ATPase activities

Auxin is an important plant growth regulator, and plays a key role in apical–basal axis formation and embryo differentiation, but the mechanism remains unclear. The level of indole-3-acetic acid (IAA) during zygote and embryo development of Nicotiana tabacum L. is investigated here using the techniques of GC-SIM-MS analysis, immunolocalization, and the GUS activity assay of DR5::GUS transgenic plants. The distribution of ABP1 and PM H+-ATPase was also detected by immunolocalization, and this is the first time that integral information has been obtained about their distribution in the zygote and in embryo development. The results showed an increase in IAA content in ovules and the polar distribution of IAA, ABP1, and PM H+-ATPase in the zygote and embryo, specifically in the top and basal parts of the embryo proper (EP) during proembryo development. For information about the regulation mechanism of auxin, an auxin transport inhibitor TIBA (2,3,5-triiodobenzoic acid) and exogenous IAA were, respectively, added to the medium for the culture of ovules at the zygote and early proembryo stages. Treatment with a suitable IAA concentration promoted zygote division and embryo differentiation, while TIBA treatment obviously suppressed these processes and caused the formation of abnormal embryos. The distribution patterns of IAA, ABP1, and PM H+-ATPase were also disturbed in the abnormal embryos. These results indicate that the polar distribution and transport of IAA begins at the zygote stage, and affects zygote division and embryo differentiation in tobacco. Moreover, ABP1 and PM H+-ATPase may play roles in zygote and embryo development and may also be involved in IAA signalling transduction

Effects of Anacetrapib in Patients with Atherosclerotic Vascular Disease

BACKGROUND: Patients with atherosclerotic vascular disease remain at high risk for cardiovascular events despite effective statin-based treatment of low-density lipoprotein (LDL) cholesterol levels. The inhibition of cholesteryl ester transfer protein (CETP) by anacetrapib reduces LDL cholesterol levels and increases high-density lipoprotein (HDL) cholesterol levels. However, trials of other CETP inhibitors have shown neutral or adverse effects on cardiovascular outcomes. METHODS: We conducted a randomized, double-blind, placebo-controlled trial involving 30,449 adults with atherosclerotic vascular disease who were receiving intensive atorvastatin therapy and who had a mean LDL cholesterol level of 61 mg per deciliter (1.58 mmol per liter), a mean non-HDL cholesterol level of 92 mg per deciliter (2.38 mmol per liter), and a mean HDL cholesterol level of 40 mg per deciliter (1.03 mmol per liter). The patients were assigned to receive either 100 mg of anacetrapib once daily (15,225 patients) or matching placebo (15,224 patients). The primary outcome was the first major coronary event, a composite of coronary death, myocardial infarction, or coronary revascularization. RESULTS: During the median follow-up period of 4.1 years, the primary outcome occurred in significantly fewer patients in the anacetrapib group than in the placebo group (1640 of 15,225 patients [10.8%] vs. 1803 of 15,224 patients [11.8%]; rate ratio, 0.91; 95% confidence interval, 0.85 to 0.97; P=0.004). The relative difference in risk was similar across multiple prespecified subgroups. At the trial midpoint, the mean level of HDL cholesterol was higher by 43 mg per deciliter (1.12 mmol per liter) in the anacetrapib group than in the placebo group (a relative difference of 104%), and the mean level of non-HDL cholesterol was lower by 17 mg per deciliter (0.44 mmol per liter), a relative difference of -18%. There were no significant between-group differences in the risk of death, cancer, or other serious adverse events. CONCLUSIONS: Among patients with atherosclerotic vascular disease who were receiving intensive statin therapy, the use of anacetrapib resulted in a lower incidence of major coronary events than the use of placebo. (Funded by Merck and others; Current Controlled Trials number, ISRCTN48678192 ; ClinicalTrials.gov number, NCT01252953 ; and EudraCT number, 2010-023467-18 .)

Virus-induced over-expression of heterologous FT for efficient speed breeding in tomato.

Potato Virus X (PVX) vectors expressing the Arabidopsis thaliana FT or tomato FT ortholog SINGLE-FLOWER TRUSS (SFT) could shorten the generation time in tomato due to accelerated tomato flowering and ripening by 14-21d and a 2-3 fold increase in the number of flowers and fruit compared with non-infected or empty vector-infected plants. The Arabidopsis FT was more effective than the tomato orthologue SFT and there was no alteration of the flower or fruit morphology. The virus is not transmitted to the next generation and viral vectors with expression of a heterologous FT will be a useful approach to speed breeding in tomato and other species.This work is supported by grants from the Biological and Biotechnology Research Council grant (BB/R018529/1) to D.C.B, and the National Natural Science Foundation of China (31972420) to Y.D

A Conserved, Serine-Rich Protein Plays Opposite Roles in N-Mediated Immunity against TMV and N-Triggered Cell Death

Plant nucleotide-binding, leucine-rich, repeat-containing proteins (NLRs) play important roles in plant immunity. NLR expression and function are tightly regulated by multiple mechanisms. In this study, a conserved serine/arginine-rich protein (SR protein) was identified through the yeast one-hybrid screening of a tobacco cDNA library using DNA fragments from the N gene, an NLR that confers immunity to tobacco mosaic virus (TMV). This SR protein showed an interaction with a 3′ genomic regulatory sequence (GRS) and has a potential role in regulating the alternative splicing of N. Thus, it was named SR regulator for N, abbreviated SR4N. Further study showed that SR4N plays a positive role in N-mediated cell death but a negative role in N protein accumulation. SR4N also promotes multiple virus replications in co-expression experiments, and this enhancement may not function through RNA silencing suppression, as it did not enhance 35S-GFP expression in co-infiltration experiments. Bioinformatic and molecular studies revealed that SR4N belongs to the SR2Z subtype of the SR protein family, which was conserved in both dicots and monocots, and its roles in repressing viral immunity and triggering cell death were also conserved. Our study revealed new roles for SR2Z family proteins in plant immunity against viruses

A role for small RNA in regulating innate immunity during plant growth

<div><p>Plant genomes encode large numbers of nucleotide-binding (NB) leucine-rich repeat (LRR) immune receptors (<i>NLR</i>) that mediate effector triggered immunity (ETI) and play key roles in protecting crops from diseases caused by devastating pathogens. Fitness costs are associated with plant <i>NLR</i> genes and regulation of <i>NLR</i> genes by micro(mi)RNAs and phased small interfering RNAs (phasiRNA) is proposed as a mechanism for reducing these fitness costs. However, whether <i>NLR</i> expression and <i>NLR</i>-mediated immunity are regulated during plant growth is unclear. We conducted genome-wide transcriptome analysis and showed that <i>NLR</i> expression gradually increased while expression of their regulatory small RNAs (sRNA) gradually decreased as plants matured, indicating that sRNAs could play a role in regulating <i>NLR</i> expression during plant growth. We further tested the role of miRNA in the growth regulation of <i>NLRs</i> using the tobacco mosaic virus (TMV) resistance gene <i>N</i>, which was targeted by miR6019 and miR6020. We showed that <i>N</i>-mediated resistance to TMV effectively restricted this virus to the infected leaves of 6-week old plants, whereas TMV infection was lethal in 1- and 3-week old seedlings due to virus-induced systemic necrosis. We further found that <i>N</i> transcript levels gradually increased while miR6019 levels gradually decreased during seedling maturation that occurs in the weeks after germination. Analyses of reporter genes in transgenic plants showed that growth regulation of <i>N</i> expression was post-transcriptionally mediated by <i>MIR6019/6020</i> whereas <i>MIR6019/6020</i> was regulated at the transcriptional level during plant growth. TMV infection of <i>MIR6019/6020</i> transgenic plants indicated a key role for miR6019-triggered phasiRNA production for regulation of <i>N</i>-mediated immunity. Together our results demonstrate a mechanistic role for miRNAs in regulating innate immunity during plant growth.</p></div

miR6019/6020 family is conserved in the Solanaceae plant family.

<p>(A) Relative <i>N</i> transcript levels in tomato D51 (blue) and VF36 (red) plants at 1-, 3- and 6 WAG stages as determined by qRT-PCR. (B) Mature miR6019 and miR6020 sequences from different <i>Solanaceae</i> plants are paired to the TIR coding sequences of the <i>N</i> gene. The sequence logo of the TIR amino acid sequences encoded by the miR6019/6020 target sequence is shown. <i>N</i> sequences highlighted in red represent the binding sites for the miR6019 and miR6020 seed sequences. Polymorphic nucleotides in mature miR6020 are highlighted in red. (C) Secondary structures of the <i>N</i>. <i>benthamiana</i> and <i>S</i>. <i>lycopersicum</i> miR6019/6020 precursors. Mature miRNA and miR star are highlighted in red. (D) Levels of tomato miR6020 and secondary siRNAs derived from the coding region of <i>N</i> at 1-, 3- and 6 WAG stages. (E) Levels of tobacco secondary siRNAs derived from the coding region of <i>N</i> at 1-, 3- and 6 WAG stages. The data are the means of three replicates with SD. Different letters indicate significant differences between the treatments according to one-way ANOVA test (P < 0.05). The Y axes in A indicate relative fold. The Y axes in D and E are in TPM units.</p

Regulation of <i>N</i>-gene-mediated resistance to TMV during D51 tomato growth.

<p>(A-C) Untreated plants at 1-, 3- and 6 WAG stages. (D-F) Plants inoculated with TMV at 1-, 3- and 6 WAG stages and photographed at 7 DPI. Arrows show the HR on the inoculated leaves. The boxed area represents enlargement of the SHR area in the systemic leaves. (G-I) Plants inoculated with TMV at 1-, 3- and 6 WAG and photographed at 21 DPI. The length of bars is labeled on each photo. (J) The average percentage rate of surviving D51 plants at different time post TMV infection with the survival rates of untreated plants as controls. The plants were infected at 1-, 3- and 6 WAG as indicated. Three independent replicates were performed. The total number (n) of plants is shown on the line chart. Y axes are in percentage units.</p

nta-miR6019/6020 regulates <i>N</i> gene expression and function during seedling growth.

<p>(A) Maps of <i>N-CFP</i>^<i>T2T1</i> and <i>N-CFP</i>^<i>t2t1</i> constructs. The sequences of wild type and mutated miR6019/miR6020 binding sites are shown above and below the map. (B) Relative CFP transcript levels measured by RT-qPCR at 1-, 3- and 6 WAG in <i>N-CFP</i>^<i>T2T1</i> and <i>N-CFP</i>^<i>t2t1</i> transgenic plants. The <i>GAPDH</i> gene was used as the reference gene. CFP levels in 1 WAG <i>N-CFP</i>^<i>T2T1</i> is considered 1. (C) Secondary structures of wild type and AXC mutants of nta-MIR6019. Mature miRNA and miRNA star sequences are highlighted in red. (D) Northern blot hybridization of sRNAs isolated from <i>nta-MIR6019</i>^<i>WT</i><i>/NN</i> and <i>nta-MIR6019</i>^<i>AXC</i><i>/NN</i> transgenic plants and both TG34 and SR1 plants at 6 WAG. Small RNA sizes are indicated to the right. (E) Relative <i>N</i> transcript levels in <i>nta-MIR6019</i>^<i>WT</i><i>/NN</i> and <i>nta-MIR6019</i>^<i>AXC</i><i>/NN</i> plants, and both TG34 and SR1 seedlings at 6 WAG. The <i>GAPDH</i> gene was used as the reference gene. The lowest <i>N</i> transcript level in <i>N</i>-containing plants (<i>nta-MIR6019</i>^<i>WT</i><i>/NN-3</i>) is considered as 1. (F) <i>nta-MIR6019</i>^<i>WT</i><i>/NN</i> and <i>nta-MIR6019</i>^<i>AXC</i><i>/NN</i> transgenic plants and both TG34 and SR1 plants inoculated with TMV at 6 WAG. The total number (n) of test plants is indicated on the image. Bars = 1cm. The data are the means of three replicates with SD. Different letters indicate significant differences between the treatments according to one-way ANOVA test (P < 0.05). Y axes indicate relative fold change.</p

Expression of nta-miR6019/6020 decreases during tobacco seedling growth.

<p>(A) Northern blot hybridization of sRNAs isolated from TG34 plants at 1-, 3- and 6 WAG. Hybridization probes, miR6019, miR156, miR168 and miR166 are indicated to the left and probe sequences are listed in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006756#ppat.1006756.s009" target="_blank">S3 Table</a>. (B) Maps of the <i>nta-MIR6019/6020a</i> promoter reporter <i>a-3kpro</i>::<i>GUS</i> and <i>nta-MIR6019/6020b</i> promoter reporter <i>b-2kpro</i>::<i>GUS</i> constructs. Open arrows represent the a-3k or b-2k promoter, and blue boxes represent GUS CDS. (C) GUS staining of <i>a-3kpro</i>::<i>GUS</i> and <i>b-2kpro</i>::<i>GUS</i> transgenic plants and SR1 wild-type plants at 1-, 3- and 6 WAG. The number (n) of stained plants and the length of each bar are indicated on the image. (D) Intensity of GUS staining in <i>a-3kpro</i>::<i>GUS</i> and <i>b-2kpro</i>::<i>GUS</i> transgenic seedlings at 1, 3 and 6WAG. GUS activity was quantified using the average gray values that are plotted on the Y-axis. (E) Relative GUS mRNA levels determined by qRT-PCR in <i>a-3kpro</i>::<i>GUS</i> and <i>b-2kpro</i>::<i>GUS</i> transgenic seedlings at 1-, 3- and 6WAG. The data are the means of three replicates with SD. Different letters indicate significant differences between the treatments according to a one-way ANOVA test (P < 0.05). Y axes all indicate relative fold.</p

<i>N</i>-mediated resistance to TMV is regulated during TG34 seedling growth.

<p>(A-C) Untreated TG34 seedlings at 1, 3 and 6 WAG, which are labeled 1W, 3W and 6W, respectively in all of the figures. (D-F) TMV-infected 1-, 3- and 6 WAG seedlings at 2 DPI. Red arrows show HR on the infected leaves. The boxed area shows an enlargement of the HR area. (G-I) TMV-infected 1-, 3- and 6 WAG seedlings at 7 DPI. (J) TMV-infected 6 WAG plant at 21 DPI. The length of bars is indicated in each photo. (K) The average percentage rate of surviving TG34 plants at different times post TMV infection with the survival rates of untreated plants as controls. The plants were infected at 1, 3 and 6 WAG as indicated. Three independent experiments were performed, and the number (n) of test plants in each experiment are shown on the graph. The Y axes are in percentage units.</p